The present invention relates to the imaging arts. It finds particular application in conjunction with low and high-density cell detection in blood smears, biological assays, and the like, and will be described with particular reference thereto. However, it is to be appreciated that the present invention will also find application in imaging other types of low-or high-density features on various substantially planar surfaces and samples, such as imaging semiconductor wafers, imaging particulate contaminants in fluids or thin solid films, and so forth, with such imaging finding specific uses in the printing arts, electronic arts, medical arts, and other scientific and engineering areas.
With particular attention to cell detection, it has been determined by the inventors that a beneficial aspect to which the present application may be applied is to scan a large number of cells, which has been considered to be in the range of 1 to 10 million cells. However, researchers and medical personnel are interested in being able to scan larger numbers of cells, such as up to 50 million or more cells, at a time. Thus, a system which can efficiently and quickly scan these large numbers of cells would be beneficial. Thereafter, the concepts of the application may be used to identify and/or locate either a small number of these cells, such as rare cells found in cancer, etc., or to be able to characterize each one of the scanned cells for use in diagnostic and/or research applications. As a general concept, the bloodstream of a living being primarily includes circulatory cells that move blood through the circulatory system, without becoming attached to other cells. However, circulatory systems also include non-circulating adherent type cells that have a tendency to adhere to markers added to the samples. For most cases, the cells being investigated are non-circulatory cells with characteristics different from circulatory cells. Once these cells are located, they may be investigated to identify their state, be counted, analyzed and/or made part of an immuno-assay (i.e., to look for proteins).
Clinical prenatal care benefits from directly accessing fetal tissues. In conventional amniocentesis, amniotic fluid surrounding the fetus is directly accessed and drawn. The amniotic fluid includes fetal cells that are extracted for study. To reduce risk to the fetus, ultrasound monitoring is typically performed during the amniocentesis to ensure that the probe needle does not contact or interfere with the fetus, and the amniocentesis procedure is performed by skilled clinical personnel. Nonetheless, amniocentesis is known to increase the risk of miscarriage with some statistics showing the risk at one in a hundred.
As an alternative to amniocentesis, rare fetal cells in the maternal bloodstream can be extracted. It is known in the prenatal medical arts that fetal cells cross the placental barrier and enter the maternal bloodstream. The concentration of fetal cells in the maternal bloodstream is typically on the order of one fetal cell for every one million maternal cells. Such “rare” fetal cells can be extracted by drawing maternal blood or by other fluid extraction. DNA analysis, fetal blood typing, or other clinical studies are performed on the rare fetal cells to provide information about the fetus. Unlike amniocentesis, extraction of rare fetal cells from the maternal bloodstream is isolated from the fetus and the womb, and the extraction can be performed by a broad range of medical personnel authorized to draw blood. In this situation the risks attendant with amniocentesis is avoided.
While the above subject matter has referred to obtaining samples from the bloodstream, detection and locating cells of different bodily fluids, such as urine, bone marrow or others, may also be beneficial.
In the clinical oncology arts, it is recognized that tumors cells are typically present in small concentrations in a patient's bloodstream. In the case of deep malignant tumors which are inaccessible except by invasive surgery, as well as tumors at different stages of development, tumor cells extracted from blood or other body fluid provide a convenient and cost effective pathway for detecting a cancer, periodically monitoring cancer remission, and diagnosing a cancer type and/or stage and monitoring treatment. Rare cell analysis targeting timorous cells is a promising diagnostic and monitoring technique for many types of cancers, including breast, lung, colon, and prostate cancers. Another beneficial aspect of rare cell research is the potential for early cancer detection prior to the formation of tumors.
In these and other rare cell studies, a problem arises because the concentration of the rare cells in the blood or other body fluid is typically very low. In a typical rare cell study, blood is processed to remove cells that that are not needed. Then a fluorescent material is applied that attaches to antibodies, which in turn selectively attach to a cell surface or cellular protein of the rare cells. The cellular proteins may be membrane proteins or proteins within a cell, such as cytoplasm proteins. The antibodies may also attach to other types of molecules of the rare cell, as well as to DNA.
The fluorescent material may be a fluorescent marker dye or any other suitable material which will identify the cells of interest. A smear treated in this manner, which may include the blood and/or components of the blood, is prepared and optically analyzed to identify rare cells of the targeted type. For statistical accuracy it is important to obtain as large a number of cells as required for a particular process, in some studies at least ten rare cells should be identified, requiring a sampling of at least ten million cells, and up to fifty million or more, for a one-in-one-million rare cell concentration. Such a blood smear typically occupies an area of about 100 cm2. It is to be understood, however, that this is simply one example and other numbers of cells may be required for statistical accuracy for a particular test or study. Other cell identifiers which are being used and investigated are quantum dots and nano-particle probes. Also, while a rare cell is mentioned as a one-in-one-million cell concentration, this is not intended to be limiting and is only given as an example of the rarity of the cells being sought. The concepts discussed herein are to be understood to be useful in higher or lower levels of cell concentration.
Turning to research applications, the scanning of a large number of cells and the characterization of each of the scanned cells may also have substantial benefits. For example, a hundred different patches, each containing 10,000 cells, maybe generated where each patch will receive a different protocol or process. Thereafter it may be useful to determine how each cell on a specific patch is affected by the protocol or process which it has undergone. One procedure of achieving such detection would be to apply a fluorescent material, and to identify those cells to which the material has become attached either to the cell's surface, cellular proteins or other portions of the cell.
A particular area of research which may benefit from the present concepts includes HIV research, where it is known the virus enters into a cell causing the cell to produce the viral protein on its membrane. However, the produced viral protein exists in very small amounts, and therefore it is difficult to detect affected cells with existing technology.
A number of cell detection methods and processes have been proposed. These include various types of automated microscopic imaging, such as described by Bauer et al. in “Reliable and Sensitive Analysis of Occult Bone Marrow Metastases Using Automated Cellular Imaging,” Clinical Cancer Researcher, Vol. 6, 3552-3559, September 2000. By use of this technique, a scan rate of approximately 500,000 cells in eighteen minutes was obtained.
Another technique used for cell detection in the blood is the use of immunomagnetic cell enrichment in combination with digital microscopy. This technique is reported by Witzig et al. in “Detection of Circulating Cytokeratin-Positive Cells in the Blood of Breast Cancer Patients Using Immunomagnetic Enrichment and Digital Microscopy”, Clinical Cancer Researcher, Vol. 8, 1085-1091, May 2002.
A proposed cancer detection technique uses reverse transcriptase polymerase chain reaction (RT-PCR) with some immunomagnetic isolation. A discussion of such a technique is, for example, set forth in the article by Ghossein et al. entitled “Molecular Detection and Characterization of Circulating Tumour Cells and Micrometastases in Solid Tumours,” European Journal of Cancer, 36 (2000) 1681-1694. Another form of immunomagnetic detection is described by Flatmark et al. in the article, “Immunomagnetic Detection of Micrometastatic Cells in Bone Marrow of Colorectal Cancer Patients,” Clinical Cancer Researcher, Vol. 8, 444-449, February 2002.
Accurate quantification of disseminated tumor cells is proposed to be obtained by using a fluorescence image analysis as disclosed by Mëhes et al. in the article entitled “Quantitative Analysis of Disseminated Tumor Cells in the Bone Marrow of Automated Fluorescence Image Analysis,” in Cytometry (Communications in Clinical Cytometry), 42:357-362 (2000). Another technique which enables a subsequent immunological characterization of isolated cells is obtained by the use of a immunomagnetic microbead isolation technique as discussed in the article by Werther et al., “The Use of the SELLection Kit™ in the Isolation of Carcinoma Cells from Mononuclear Cell Suppression,” Journal of Immunological Methods, 238 (2000) 133-141.
Burchill et al. provides a review and comparison of several detection methods in “Comparison of the RNA-amplification Based Methods RT-PCR and NASBA for the Detection of Circulating Tumour Cells,” British Journal of Cancer, (2002) 86, 102-109. Discussed are studies which suggest nucleic acid sequence-based amplification (NASBA) of targeted RNA may provide a robust manner of detecting cancer cells.
The above papers illustrate the wide range of research which is being undertaken in the are of rare cell detection and identification. In this regard, the ability to scan large numbers of cells at a high rate is considered a key aspect which increases the throughput of the testing processes. The processes described in the cited papers set forth a variety of cell detection and location techniques. It is considered to be valuable to provide a system which improves the speed, reliability and processing costs which may be achieved by the systems or processes cited in the above papers.
A cell detection technique which is noted in more specific detail is fluorescence in situ hybridization (FISH). This process uses fluorescent molecules to paint genes or chromosomes. The technique is particularly useful for gene mapping and for identifying chromosomal abnormalities. In the FISH process, short sequences of single-stranded DNA, called probes, are prepared and which are complementary to the DNA sequences which are to be painted and examined. These probes hybridize, or bind, to a complementary DNA, and as they are labeled with a fluorescent tag, it permits a researcher to identify the location of sequences of the DNA. The FISH technique may be performed on non-dividing cells.
Another process of cell detection is flow cytometry (FC), which is a means of measuring certain physical and chemical characteristics of cells or particles as they travel in suspension past a sensing point. Ideally the cells travel past the sensing point one by one. However, significant obstacles exist to achieving this ideal performance, and in practice a statistically relevant number of cells are not detected due to the cells bunching or clumping together, making it not possible to identify each cell individually. In operation a light source emits light to collection optics, and electronics with a computer translates signals to data. Many flow cytometers have the ability to sort, or physically separate particles of interest, from a sample.
Another cytometry process is known as laser scanning cytometry (LSC). In this system, data is collected by rastering a laser beam within the limited field of view (FOV) of a microscope. With laser rastering, the excitation is intense and in a single wavelength, which permits a differentiation between dyes responsive at distinct wavelengths. This method provides equivalent data of a flow cytometer, but is a slide based system. It permits light scatter and fluorescence, but also records the position of each measurement. By this design, cells of interest can be relocated, visualized, restained, remeasured and photographed.
While it is appreciated that increasing the speed at which cells are scanned is a valuable characteristic, a problem with these existing cell analysis techniques is the use of conventional technology which have relatively small fields of view (FOV), such as microscopes. To overcome the FOV limitation, cell analyses often employ automated high-speed scanning which however produces substantial undesirable acceleration forces on the scanned stage.
Another problem in cell studies, for both high and low density situations, is that the fluorescence intensity produced by treated cells is low, around 1000-2000 flours (fluorescent molecules). A high numerical aperture for the light-collecting aperture is preferred in the optical analysis system to provide good light collection.
Yet another problem in the cell studies is resolution. For example, if a cell has a diameter of about ten microns, the optics for the cell analysis preferably provides a resolution of this order. However, achieving high resolution typically requires a reduced field of view and consequently results in a decreased scanning speed and increased required sampling time.
The present invention contemplates a new and improved apparatus and method which overcomes the above-referenced problems and others.